Cellulases (.beta.-1,4-endoglucanase) are a family of enzymes that work together to break down cellulose to its simple sugar components. Cellulose may also be degraded via acid hydrolysis using much harsher conditions than required by cellulase enzymes. Furthermore, cellulases catalyze highly specific reactions, yeilding specific products, and are required in much smaller quantities compared to acid hydrolysis reactions.
Cellulose degrading enzymes are used for a wide variety of industrial applications. One of the major potential uses of cellulase is in the conversion of cellulosic biomass to industrially important end products (ie; sugars, which can be fermented to produce a variety of products). For example, production of fuel ethanol is typically produced from grains such as corn. A similar process utilizing high cellulosic rice straws is currently under development. Unfortunately, ethanol produced by such methods is still too expensive to compete commercially with gasoline. However, improvements in technology to utilize wood, grass and other high cellulose containing biomass for the production of ethanol would be valuable to the art for production of a less expensive and cleaner fuel source.
In addition to biomass coversion, cellulose degrading enzymes find utility in a variety of other industrial products and processes including: textile finishing, production of detergent additives, food and beverage processing, feed additives, ensiling and fermentation processes.
Current methods for the production of cellulose degrading enzymes are generally believed to be limiting to the further development of a lignocellulosic ethanol industry. Filamentous fungi are well known for the production of industrial cellulases. However, economical production of cellulase is compounded by the relatively slow growth rates of cellulase producing fungi, the long times required for cellulase induction an the high value of the product ethanol.
Recently, genes encoding cellulose degrading enzymes have been cloned from a variety of cellulytic bacteria and fungi. Cloned genes encoding cellulases having very high specific activities over a broad pH range in addition to high thermostability are considered most desirable for bioethanol derived processes.
Recombinant bacterial or fungal hosts producing cellulose degrading enzymes have been the focus of recent efforts for the production of various cellulase preparations. However, production of cellulases in plants may find use in the art.
Since one of the major components of plants is cellulose, it would be expected that the production of cellulose degrading enzymes in plants cells may have detrimental effects to the host organism. However, by compartmentalizing the expressed cellulose degrading enzyme in a plant organelle, for example in a plastid, any detrimental effects of cellulase enzyme expression may be overcome. Furthermore, utilization of a cellulose degrading enzyme with a high temperature and/or pH optimum may also provide safeguards for the expression of such enzymes in plants that are grown at ambient temperatures.
Plant plastids (chloroplasts, amyloplasts, elaioplasts, chromoplasts, etc.) are the major biosynthetic centers that, in addition to photosynthesis, are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. Plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which potentially can result in very high levels of foreign gene expression. In addition, plastids of most plants are maternally inherited. Consequently, heterologous genes expressed in plastids are not pollen disseminated, therefore, a trait introduced into a plant plastid will not be transmitted to wild-type relatives by cross-fertilization. Thus, the plastids of higher plants are an attractive target for genetic engineering.
Several plastid expression systems have been developed utilizing regulatory elements derived from genes highly expressed in plant plastids. For example, promoters commonly employed to express genes in plastids are derived from the promoter regions of the 16S ribosomal RNA operon (Prrn), from the promoter region of the gene encoding for a core protein of photosystem II, the D1thylakoid membrane protein (PpsbA), or from the promoter region of the ribulose 1,5-bisphosphate carboxylase gene (PrbcL).
In addition, a totally heterologous expression system has been developed to express DNA sequences in plant plastids (McBride et al U.S. Pat. No. 5,576,198, the entirety of which is incorporated herein by reference). This system is a two component system. The first component is a plastid transgene driven by a T7 bacteriophage gene 10 promoter/leader sequence. The second component is a nuclear gene encoding the T7 RNA polymerase that is targeted to the plastid compartment. This two component expression system allows for the controlled, high level expression of DNA sequences in the plant plastid.
Utilizing high-level plastid expression offers an attractive opportunity for the expression of industrial proteins, such as thermophilic cellulases and related thermophilic polysaccharide hydrolyzing enzymes (i.e., cellobiohydrolase, xylanase, hemicellulase) in plant plastids. Expression of such enzymes in plant plastids provides an alternative source for the production of polysaccharide degrading enzymes utilized for industrial products/processes (textile finishing, detergents, food and beverage processing, feed additives, ensiling, pulping, paper making, and biomass conversions). Also, the expression of thermophilic cellulases and related cellulose degrading enzymes in plant plastids provides an alternative or supplementary method for degrading endogenous cellulose contained in plant tissues and releasing the stored carbon (as monosaccharides) for subsequent fermentative processes. In addition, plastid sequestration (isolation from the substrate)and the high temperature optimum (versus low activity at ambient temperatures)of the thermophilic cellulase provide two internal safeguards for protecting the plant from the intrinsic enzyme activity during critical plant growth and development stages.
Thus, expression of thermophilic cellulose degrading enzymes in plant plastids offers the opportunity for an less expensive and abundant source of cellulose degrading enzymes.